Functional analysis of Bicc1 and novel interacting proteins in renal tubule cell metabolism and polycystic kidneys

Genetic disorders known as ciliopathies develop polycystic kidneys, including autosomal dominant and autosomal recessive polycystic kidney diseases (ADPKD and ARPKD). Several signaling pathways including the cAMP/PKA pathway are implicated in driving cystic growth. However, the mechanisms underlying cysts formation is still elusive. Renal cysts also arise in patients and in mouse models with mutations in the RNA-binding protein Bicaudal-C (Bicc1). Bicc1 binds specific mRNAs such as adenylate cyclase VI (AC6) mRNA for translational repression. Conducting a structure-function analysis, we demonstrated that Bicc1 self-polymerizes through its sterile alpha motif (SAM domain) to form cytoplasmic clusters, which specifically localize and silence bound mRNAs. Altered glucose and lipid metabolism also sustain cystic growth in polycystic kidneys, but the cause of these perturbations is unclear. Here, we found that Bicc1 is essential for normal expression of the gluconeogenic enzymes FBP1 and PEPCK specifically in kidneys, and to maintain euglycemia. In a proteomic interactome screen using a knock-in cell line, we found that TAP-tagged Bicc1 binds the C-Terminal to Lis-Homology domain (CTLH) complex, the mammalian orthologue of the glucose-induced degradation (Gid) complex that triggers degradation of FBP1 in S. cerevisiae. Bicc1 stimulates FBP1 expression in vitro in cultured murine inner medullary collecting duct (mIMCD3) cells, albeit independently of its interaction with the CTLH complex. Instead, Bicc1 and CTLH complex seem to mutually downregulate each other. Bicc1 interactome is also enriched with metabolic related proteins. Here we found that Bicc1 coimmunoprecipitates with rate-limiting metabolic enzymes and contributes to maintain their protein levels, and that deletion of Bicc1 significantly alters the renal metabolome while enhances autophagy. Altogether, these results implicate Bicc1 as an important metabolic regulator in the etiology of polycystic kidneys.

Regulation of the mRNA silencing activity of Bicaudal-C

Florian Urs Bernet

Cilia dysfunction is a common factor underlying left-right axis malformation and the pathogenesis of virtually all known renal cystic diseases. Mutations in the RNA-binding protein Bicaudal-C (Bicc1) provoke cystic kidneys reminiscent of polycystic kidney disease (PKD), and ectopic Wnt/b-catenin signaling during left-right axis development. Renal cysts in these mice have been linked to defective miRNA-mediated silencing of specific mRNAs, including adenylate cyclase 6 (AC6) and protein kinase A inhibitor (PKIa), but little is known about how Bicc1 activities are regulated. Prompted by the overlapping phenotypes of mice lacking Bicc1 or Inversin (Invs), the protein defective in patients with nephronophthisis type II, we investigated a potential epistatic relationship. We found that Invs acts as an inhibitor of Bicc1 induced silencing and that Invs is required for Bicc1 localization to the Invs compartmentent in the proximal cilium. Molecular analysis demonstrated that Invs interacts with the Bicc1 KH domains required for mRNA binding. The Bicc1/Invs complex prevented the recruitment of target mRNA and their incorporation into miRNA-induced silencing complexes (miRISC). To test whether Bicc1 activity is regulated in vivo, we monitored Bicc1 targets in inv/inv mutant mouse kidneys lacking Invs. While AC6, the RNA helicase Ddx5 (a novel Bicc1 target) and PKA signaling were dramatically downregulated in inv/inv mutant kidneys, these effects were suppressed in Bicc1-/-; inv/inv double mutants, demonstrating that loss of Invs leads to ectopic activation of Bicc1. In autosomal dominant polycystic kidney disease (ADPKD), fluid-filled cysts develop in both kidneys due to defective calcium-permeable mechanosensory complexes of polycystin-1 and -2 encoded by PKD1 and PKD2. Studies in zebrafish suggest that Pkd2/Ca2+ signaling stimulates calmodulin kinase 2 (CaMK2). To further elucidate a role for Ca2+ in Bicc1 regulation, we mutated specific residues that are predicted to be phosphorylated by CaMK2. We found that CaMK2 phosphorylated Bicc1 on T724 and S805 and enabled miRISC assembly, at least in part by stimulating Bicc1 binding to nascent miRNAs. In addition, elevated Ca2+ concentrations prevented in vitro binding of Bicc1 to Invs. Based on these data we suggest a dual role for Ca2+ in regulating Bicc1. Elevated intracellular Ca2+ levels may induce the dissociation of a Bicc1/Invs complex and activate CaMK2 to enable Bicc1 mediated silencing of target mRNAs.

EPFL2015

Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing

Daniel Constam, Charlotte Maisonneuve

Genetic defects in autosomal dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are upregulated in cystic kidneys of Bicc1-/- knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. Repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules

Oxford Univ Press2012

Quantitative single cell dynamics of signaling and transcriptional response in mammalian cells

Onur Tidin

Cells live in ever-changing environments, thereby facing a variety of dynamic environmental signals. Environmental stimuli elicit intracellular responses through signaling pathways, which converge on transcriptional activation or repression of target genes. Despite intensive research, dissecting the complex interactions between pathway components that modulate mRNA and protein production still remains a difficult task. To this end, single-cell approaches provide unique insights into intracellular processes and cell responses to environmental stimuli, otherwise inaccessible with traditional bulk studies. Single-cell measurements have revealed that isogenic cells sharing the same environment display substantial heterogeneity in both transcript and protein level. As the initial step in gene expression, transcription is an important source of such variability in eukaryotic cells due to low number of molecules involved, transcription factor dynamics and the discrete nature of biochemical reactions. Notably, production of mRNA during transcription occurs in short periods of activity, termed as transcriptional bursts, followed by longer periods of inactivity. Despite widespread observations of transcriptional dynamics in mammals, upstream molecular mechanisms shaping the eukaryotic transcription remained elusive. In this study, we quantitatively linked upstream factors and transcriptional kinetics by developing an experimental system to temporally monitor, simultaneously, inputs (transcription factors) and outputs (target gene expression) in mammalian cells, in response to stimulus. We established two Tet-On inducible stable cell lines each expressing a fusion protein of a luminescence (Nanoluciferase) reporter to either SMAD4 or SMAD2, the two main transcrip- tion factors downstream of the TGF-Î² pathway. These cell lines that also contain a short-lived firefly luciferase reporter for the expression of the target endogenous connective tissue growth factor (ctgf ) gene allowed us to quantitatively link nuclear accumulation of SMADs upon TGF-Î² stimulation to the target gene expression in real-time single cell measurements. Time- lapse luminescence microscopy and image analysis with the custom developed CAST (Cell Automated Segmentation and Tracking platform) platform provided quantitative single-cell data to link the upstream transcription factor profile to its target gene activity. The data revealed weak single-cell correlations between translocation level and the target gene expression response which suggests a mechanism in which the translocation of SMADs initiates the transcriptional response but affects the response amplitude minimally. However, SMAD4 expression levels influenced the target gene response dynamics such that cells with high SMAD4 abundance favored to respond in a more sustained and even oscillatory manner. This suggests a mechanism consisting of a dynamic interplay between the transcriptional activators and feedback mechanisms in TGF-Î² signaling. We explored further the effect of different factors such as ligand concentration, ligand type on both signaling and target gene response. Taken together, this study proposes an experimental and quantitative framework that dissect mechanisms underlying transcriptional response to stimulus.